This proposal is to investigate the molecular basis for activity-dependent and neurotransmitter-dependent modulation of the biophysical and biochemical properties of potassium channels. We are testing the hypothesis that short-term and long-term regulation of the Kv4.2 channel is mediated by phosphorylation. Specifically we hypothesize that protein kinase-dependent regulation of Kv4.2 is mediated by direct phosphorylation of the pore-forming alpha subunit. In our studies over the last few years we have identified 9 different phosphorylation sites on Kv4.2, mediated by 4 different second-messenger-regulated kinases. Moreover, we have found that PKC phosphorylation of the Kv4.2 C-terminal cytoplasmic domain can regulate the capacity of ERK MAP Kinase to phosphorylate this same channel subregion. Addressing the mechanism for this PKC/ERK interaction will be the first Specific Aim of this project: To determine the protein structure/function relationships underlying PKC modulation of ERK phosphorylation of the C-terminal cytoplasmic domain of Kv4.2. Another example of the complexity of phosphorylationdependent regulation of Kv4.2 is illustrated by considering PKA regulation of Kv4.2. In recent studies we found that PKA regulation of Kv4.2-encoded currents required the presence of a KChlP ancillary subunit. Despite the ability of PKA to phosphorylate the Kv4.2 alpha subunit in the absence of KChlP3, PKA was unable to alter channel biophysical properties by this mechanism alone. Thus, we propose Specific Aim 2 of the project: To determine the structure/function relationships for KChlP modulation of phosphoregulation of Kv4.2. Our final Specific Aim will focus on Calcium/calmodulin-dependent Protein Kinase II (CaMKII) regulation of Kv4.2. We have identified two sites of CaMKII phosphorylation on Kv4.2 and determined that phosphorylation at one of these sites (T438) is necessary and sufficient for CaMKII stabilization and enhanced surface expression of Kv4.2 in COS cells. Additional preliminary results indicate that CaMKII regulation of Kv4.2 surface expression occurs in hippocampal pyramidal neurons as well. We will test the hypothesis that phosphorylation-dependent protein-protein interactions mediate this effect in the final Specific Aim: To investigate the mechanism of CaMKII regulation of Kv4.2 expression and protein stabilization. These studies should allow for the first time the definition of specific phosphorylation events, of known physiologic consequence, as mechanisms for Kv4.2 A-type potassium channel regulation.